Current efforts to produce biofuels using synthetic biology have focused on using model organisms (E. coli and S. cerevisiae) as chassis of metabolic engineering [1, 2]. These efforts have concentrated on using biomass-derived carbohydrates as the sources for renewable sources biofuel generation [3]. These strategies require redirection of central metabolic pathways by introduction of new pathways that redirect metabolic flux to a desired end-product. This approach has been used to produce alcohols, alkenes and isoprenoids that may be used as liquid fuel substitutes for petroleum [4]. Rewiring the metabolism of these model organisms so they can utilize CO2 as the carbon input for biofuel production would have substantial benefits in broadening the substrate scope for metabolic engineering and reducing CO2 emissions. However, transforming model organisms such as E. coli into an autotroph remains a daunting task that has not been accomplished.
One class of chemoautotrophic bacteria, “Knallgas” bacteria that grow with H2/CO2 under aerobic conditions, does not have these limitations. The model strain of this class, Ralstonia eutropha, can grow to very high cell densities (>200 g/L) and has been extensively manipulated genetically [5]. Under nutrient limitation, R. eutropha directs most of the reduced carbon flux generated by the Calvin cycle to synthesis of polyhydroxybutyrate (PHB), a biopolymeric compound stored in granules. Under growth with H2/CO2, 61 g/L of PHB was formed in 40 h, which represents ˜70% of total cell weight (FIG. 1)[6]. PHB and related polyhydroxyalkonate polymers have been produced on industrial scale and marketed as Biopol™ (Monsanto; St. Louis, Mo.) and Mircel™ (Metabolix; Cambridge, Mass.) [5]. Therefore, R. eutropha is an attractive alternative for biofuel production from CO2 as it already has the capability for autotrophic growth, is amenable to metabolic engineering and expresses a metabolic pathway that supports significant carbon flux.
An inexpensive source of H2 will be essential for the effective development of R. eutropha as a biofuel-producing platform. Known small-molecule metal catalysts generally require organic acids, additives, and/or solvents that are also incompatible for use with living organisms [7]. Traditional catalysts in this area rely on sensitive thiol or phosphine donors, a key advance in this strategy is the choice of pyridine donors as a building block for ligand design, as they support water-stable and water-soluble complexes with reasonable reduction potentials based on strong s-donor/mild p-acceptor properties [8]. By using these rugged donor groups, inexpensive catalysts containing earth-abundant metal centers are accessible that are soluble and stable in microbial growth media. R. eutropha is an ideal microbe to couple with electrocatalysis, as growth with H2 generated in situ by an electrode has already been demonstrated [9].